Spatially-defined macrocycles incorporating peptide bond surrogates

ABSTRACT

Novel spatially-defined macrocyclic compounds incorporating peptide bond surrogates are disclosed. Libraries of these macrocycles are then used to select one or more macrocycle species that exhibit a specific interaction with a particular biological target, in particular, compounds according to the invention are disclosed as agonists or antagonists of a mammalian motilin receptor and a mammalian ghrelin receptor.

RELATED APPLICATION INFORMATION

The present application is a divisional application of and claimspriority from U.S. patent application Ser. No. 10/911,219, filed Aug. 2,2004, which claims the benefit of U.S. Patent Application Ser. No.60/491,250, filed Jul. 31, 2003; U.S. Patent Application Ser. No.60/491,253, filed Jul. 31, 2003; and U.S. Patent Application Ser. No.60/491,249, filed Jul. 31, 2003. The disclosure of each of which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to spatially-defined macrocyclic compoundsincorporating peptide bond surrogates. It also relates to the generationof libraries of these macrocycles. These libraries are then used toselect one or more macrocycle species that exhibit a specificinteraction with a particular biological target.

BACKGROUND OF THE INVENTION

Peptides have been at the forefront of combinatorial chemistrytechnology development due to their ease of synthesis on solid support,the reproducible and high-yielding reactions involved, and the readyavailability of starting materials. Peptides are the endogenous ligandsfor a number of enzymes and receptors. Modifications of these peptidescan be performed to develop even more potent agonists or inhibitors ofthese same receptors and enzymes. In addition, combinatorial peptidelibraries have been used to find a number of previously unknown activesequences for a wide array of enzyme and receptor systems. However,these novel materials are still plagued by the usual limitationsassociated with the direct use of peptides as pharmaceuticals, althoughmany are used in human and veterinary medicine due to their potency andselectivity. Although peptides are highly potent and selectivebiological agents, their use as pharmaceutical products is limited by

-   -   Poor aqueous solubility    -   Metabolic instability, particularly to proteases    -   Low oral bioavailability    -   Inadequate membrane permeability    -   Difficulty in transport to site of action in tissues and organs    -   Potential antigenicity    -   Short pharmacokinetic half-life decreases duration of        pharmacological action    -   Side effects due to the presence of receptors for the peptide in        other non-target areas of an organism    -   High manufacturing costs

In order to circumvent these drawbacks while retaining the high potencyof the peptide, significant work over the past three decades has beendevoted to the study of mimics of these peptides, or peptidomimetics.Replacement of one or more amide bonds with functional groups that havesimilar structural characteristics, but different metabolic profiles hasbeen pursued widely. Similarly, restriction of conformation of theresulting molecules utilizing either sterically demanding orstructurally restricted amino acids to specifically display side chainsin space. Cyclization of the linear peptide is also traditionallypursued.

However, the ability to control conformation within a single cyclicmolecule often requires long experimentation in order to access thedesired structure. Of greater interest would be the ability to directand control the three-dimensional orientation so as to probe multipleconformations with the same interacting peptide side chainfunctionalities. In this manner, the optimal one for the biologicaltarget of interest could be rapidly determined.

Recently, WO 01/25257 has described the use of specific elements termed“tethers” to control the conformations within macrocyclicpeptidomimetics. However, to date, no method has been described tocombine the use of such tether elements with peptide bond surrogates.

Such molecules would have unique and superior properties over otheranalogues:

-   -   Ease of synthesis    -   Enhanced chemical stability    -   Improved metabolic stability    -   Better selectivity with lower incidence of side effects    -   More favorable pharmacokinetics    -   Better oral bioavailability    -   Higher aqueous solubility

In particular, these analogues possess advantages that make themdesirable as pharmaceutical agents with improved therapeutic properties:

-   -   Additional interacting functionalities    -   Modulated physicochemical properties    -   Modified conformations to those of cyclic peptides that are        dictated primarily by the amide bond

The use of backbone to backbone cyclization to control conformation ofpeptidic molecules has been described. (Gilon, G. Biopolymers 1991, 31,745). However, this approach provides a constraint with the only controlbeing provided by the length of the backbone chain employed. This doesnot permit access to all the conformations that might be require inorder to optimally interact within a biological system. Nonetheless,this approach has yielded somatostatin analogues that can be used fortherapeutic (WO 98/04583, WO 99/65508, U.S. Pat. No. 5,770,687, U.S.Pat. No. 6,051,554) or diagnostic purposes (WO 02/062819), bradykininanalogues (U.S. Pat. No. 5,874,529).

On the other hand, cyclic peptides offer a number of benefits comparedwith the corresponding linear analogues, including restrictedconformational mobility, defined topology, enhanced stability toproteolytic enzymes and modified polarity (Molecular Diversity 2000(pub. 2002), 5, 289-304).

Accordingly, cyclic structures can greatly improve the pharmacologicaland pharmacokinetic profiles of peptides. Examples demonstrate thatcyclic peptides can enhance potency, selectivity, stability,bioavailability and membrane permeability. The stability to enzymaticdegradation of the cyclic structure arises from the difficulty of suchmolecules to attain the extended conformation required to be recognizedas a substrate for peptidases. Very large mixture libraries (10⁸ membersor more) of cyclic peptides have been described in WO 98/54577.

Until recently, the number of reports of the use of macrocyclicpeptidomimetics in drug discovery has rather been limited. Recentexamples of therapeutically interesting bioactivities that have beendisplayed by small peptide or peptidomimetic macrocycles includeprotease inhibition (HIV, cancer, inflammation)—Curr. Med. Chem. 2001,8, 893-907; Integrin receptor antagonists (cell adhesion inhibition,inflammation, diabetes)—J. Med. Chem. 2001, 44, 2586-2592; Histonedeacetylase inhibition (cancer, anti-fungal)—Tr. Endocrin. Metabol.2001, 12, 294-300; Curr. Med. Chem. 2001, 8, 211-235; Urotensin IIantagonists (cardiovascular disease)—Angew. Chem. Int. Ed. 2002, 41,2940-2944; neurokinin-2 antagonists (asthma, irritable bowelsyndrome)—J. Med. Chem. 2002, 45, 3418-3429; tyrosine receptor kinase A(TrkA) antagonists and neurotrophin-3 mimetics (Alzheimer's, stroke,diabetic neuropathy)—Mol. Pharm. 2000, 57, 385-391; J. Org. Chem. 2004;69, 701-713; antibacterial agents—J. Med. Chem. 2002, 45, 3430-3439; andC5a complement inhibitors (inflammatory diseases)—Br. J. Pharmacol.1999, 128, 1461-1466.

However, in most of these cases, the formation of the cyclic structurewas simply one step in a lengthy optimization process. The use of largemacrocyclic libraries for initial hit identification and drug discoveryis largely unprecedented. This is particularly striking given theextensive efforts in combinatorial chemistry, which began focused onpeptides, and the subsequent explosion in the number and type of smallmolecule libraries that can now be accessed.

Among the possible modifications of peptide bonds, depsipeptides areknown in the art. A comparative example of a given peptide and thecorresponding depsipeptide is given below. Importantly, the relativearrangement of side chains on adjacent residues is not affected as itcan be with other peptide bond surrogates.

As may be noticed, one of the —NH— in the peptide is replaced by —O— inthe depsipeptide.

Many depsipeptides are known to exhibit special biological activities(see Ballard, C. E.; Yu, H.; Wang, B. Curr. Med. Chem. 2002, 9, 471-498;Moore, R. E. J. Ind. Microbiol. 1996, 16, 134-143 and Shemayakin, M. M.Antimicrob. Agents Chemother 1965, 5, 962-976). For example, vancomycin,valinomycin, actinomycins, didemnins, dolstatins are natural productdepsipeptides. Included in the therapeutic utility of these compoundsare anticancer, antibacterial, antiviral (callipeltins, quinoxapeptins),antifungal (jaspamides), anti-inflammatory (neurokinin antagonists),anti-clotting, antiantherogenic (micropeptins), and other activities.

Another class of amino acid mimics, peptoids, have found wide utility inthe design and synthesis of peptide-related therapeutic agents andbiomaterials (Curr. Opin. Struct. Biol. 1999, 9, 530-535). A comparisonbetween depsipeptides and peptoids is shown below:

In yet another approach, the urethane moiety can function as aneffective peptide bond mimic. It possesses analogous defined planarityand geometry with similar rigidity to that of an amide bond. However,this moiety is not isosteric to the amide as it contains an extra atom,so that incorporation leads to larger-sized structures. This could provequite advantageous, however, as the unique properties of peptidescontaining β-amino acids attests (Chem. Rev. 2001, 101, 3893-4011; Curr.Med. Chem. 2002, 9, 811-822).

The following can be cited as potential benefits of the urethane moietyas a peptide bond surrogate:

-   -   Modification of H-bonding properties due to the extra heteroatom        for inter- and intramolecular interactions as well as in        improved solubilities    -   Imposition of a degree of conformational restriction    -   Backbone NH and chiral R groups offer opportunities for        substitution and modification for modulation of biological and        physical properties    -   Modified polarity, more lipophilic due to the extra carbon atom,        as compared with the peptide bond    -   Resistance to proteinases    -   Alteration of pharmacokinetic properties

Urea peptide bond surrogates have also been explored in combination withother isosteres to construct molecules with novel architecture. Forexample, in the development of linear tripeptidomimetics as matrixmetalloproteinase inhibitors for the treatment of arthritis and cancer,ureas and sulfonamides were targeted as replacements for the amide bond.The urea substitution actually contains an N-substituent where theattached group is the same as the amino acid side chain in the originalpeptide and hence could be considered a urea-peptoid hybrid.

These examples highlight only a representative sampling of the varietyof peptide bond surrogates that have been designed and investigated(Mini-Rev. Med. Chem. 2002, 2, 463-473; Mini-Rev. Med. Chem. 2002, 2,447-462; Curr. Med. Chem. 2002, 9, 2209-2229; Curr. Med. Chem. 2002, 9,2243-2270; Curr. Med. Chem. 2002, 9, 963-978; Curr. Opin. Chem. Biol.2002, 6, 872-877; Curr. Opin. Chem. Biol. 1998, 2, 441-452; Angew. Chem.Int. Ed. Engl. 1994, 33, 1699; J. Med. Chem. 1993, 36, 3039-3049; J.Org. Chem. 2000, 65, 7667-7675). Additional structures that specificallyreplace the peptide bond or offer an alternative type of peptide residueare shown in FIG. 3. This variety has permitted chemists to explore anumber of modifications to peptide structure not accessible throughnatural amino acids alone. However, often this is done, not in apredictable manner, but rather determined after the construction of themolecule. Therefore, the control permitted by the aforementioned tetherelements would be of utility in the context of structures containingthese peptide bond surrogates.

Further, to date, peptide bond surrogates have not been widelyinvestigated in the context of cyclic structures nor in libraries,likely due to the challenges involved in their syntheses.

Accordingly, their remains a need for macrocyclic structuresincorporating a variety of peptide bond surrogates.

SUMMARY OF THE INVENTION

The present invention uses peptide bond surrogates in the context ofconformationally-defined cyclic molecules. Accordingly, the inventionrelates to macrocyclic compounds of formula (I) which incorporatepeptide bond surrogates.

whereinA₃ and A₄ are optionally present;A₁, A₂, A₃ and A₄ are chosen from the group consisting of formulas S1 toS21, with the proviso that at least one of A₁, A₂, A₃ or A₄ is selectedfrom the group consisting of formulas S2 to S21

-   -   wherein R and R′ are chosen from side chains of natural amino        acids or side chains of unnatural amino acids, with the proviso        that R′ is not hydrogen;    -   R″ is hydrogen or alkyl;    -   m is 0, 1 or 2; and    -   n is 0, 1 or 2;        X is —O— or —NR₁—, wherein R₁ is selected from the group        consisting of hydrogen, alkyl, substituted alkyl, acyl and        sulfonyl; and        T is a bivalent radical of formula (II):

—J—(CH₂)_(d)—K—(CH₂)_(e)-L-(CH₂)_(f)—  (II)

-   -   wherein:    -   J is bonded to X and is a bivalent radical chosen from —CH₂— or        —C(═O)—;    -   d, e and f are each independently selected from 0, 1, 2, 3, 4 or        5;    -   L is optionally present;    -   K and L are independently a covalent bond or a bivalent radical        selected from the group consisting of:        -   —O—, —NR₂—, —S—, —SO—, —SO₂—, —C(═O)—, —C(═O)—O—, —O—C(═OQ,            —C(═O)—NR₁, —NR₃—C(═O)—, —SO₂—NR₄—, —NR₄—SO 2-1-CR₅R₆—,            —CH(OR₇)—, —CH═CH—        -   with a Z or E configuration, —C═C—, and

-   -   -   wherein R₂ is selected from the group consisting of            hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted            cycloalkyl, heterocyclic, substituted heterocyclic, aryl,            substituted aryl, heteroaryl, substituted heteroaryl,            formyl, acyl, carboxyalkyl, carboxyaryl, amido, amidino,            sulfonyl and sulfonamido        -   R₃ and R₄ are independently selected from hydrogen or C₁-C₆            alkyl;        -   R₅ and R₆ are independently selected from the group            consisting of hydrogen, alkyl, hydroxy, alkoxy, and amino,            with the proviso that if one of R₅ or R₆ is hydroxy, alkoxy            or amino, the other is hydrogen or alkyl;        -   R₇ is selected from the group consisting of hydrogen, alkyl,            substituted alkyl, formyl and acyl; and        -   G₁ and G₂ are independently a covalent bond or a bivalent            radical selected from the group consisting of:            -   —O—, —NR₈—, —C(═O)—, —C(═O)—O—, —O—C(═O)O—, —C(═O)NR₉—,                —NR₉—C(═O)—, —SO₂—NR₁₀—, —NR₁₀—SO₂—, —CR₁₁R₁₂—, —CH═CH—                with Z or E configuration, and —C≡C—, wherein R₈ is                selected from the group consisting of hydrogen, alkyl,                substituted alkyl, cycloalkyl, substituted cycloalkyl,                heterocyclic, substituted heterocyclic, aryl,                substituted aryl, heteroaryl, substituted heteroaryl,                formyl, acyl, carboxyalkyl, carboxyaryl, amido, amidino,                sulfonyl and sulfonamido;            -   R₉ and R₁₀ are independently selected from hydrogen or                C₁-C₆ alkyl; and            -   R₁₁ and R₁₂ are independently selected from the group                consisting of hydrogen, alkyl, hydroxyl, alkoxy, and                amino with the proviso that if one of R₁₁ or R₁₂ is                hydroxyl, alkoxy or amino, the other is hydrogen or                alkyl;        -   with the proviso that G₁ is the closest to J, and that G₁ or            G₂ can be attached in a relative arrangement to each other            of 1, 2 or 1, 3 or 1, 4.

In a second aspect of the invention, there are provided compounds offormula (III)

wherein A₁, A₂, A₃, A₄ and X are as defined for formula (I) and whereinT₂ is a bivalent radical chosen from the group consisting of:

wherein the wavy lines indicate either a (R) or (S) stereochemistry ormixture thereof;q₁, q₂, q₃, q₆, q₇, q₈, q₉, q₁₀, q₁₁, q₁₃, q₁₅ and q₁₆ are eachindependently 1, 2, 3, 4 or 5;q₄ and q₁₈ are independently 1 or 2;q₅ is 2, 3 or 5;q₁₂ and q₁₄ are each independently 0, 1, 2, 3 or 4;q₁₇ is 0, 1, 2 or 3;P₁, P₂, P₃ P₄ and P₅ are each independently O, S or NH;

P₆ is N or CH; P₇ is O or CR₅₂R₅₃;

R₃₆ is hydrogen, C₁-C₆ alkyl, benzyl or acyl;R₅₀ and R₅₁ are independently selected from the group consisting ofhydrogen, alkyl, hydroxy, alkoxy, and amino with the proviso that if oneof R₅₀ or R₅₁ is hydroxy, alkoxy or amino, the other is hydrogen oralkyl;R₅₂ and R₅₃ are independently selected from the group consisting ofhydrogen, alkyl, hydroxy, alkoxy, and amino with the proviso that if oneof R₅₂ or R₅₃ is hydroxyl, alkoxy or amino, the other is hydrogen oralkyl;R₅₄, R₅₅, R₅₆, R₅₇ and R₅₈ are independently selected from the groupconsisting of hydrogen, alkyl, hydroxy, alkoxy, and amino;R_(AA) is the side chain of a natural amino acid;(X) indicates the point of attachment of T₂ to X; and(W) indicates the point of attachment of T₂ to A₂, A₃ or A₄.

The invention also provides combinatorial libraries of thesemacrocycles. Compounds of formula (I) and formula (III) are alsodisclosed as agonists or antagonists of a mammalian motilin receptor anda mammalian ghrelin receptor.

While the invention will be described in conjunction with an exampleembodiment, it will be understood that it is not intended to limit thescope of the invention to such embodiment. On the contrary, it isintended to cover all alternatives, modifications and equivalents as maybe included as defined by the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a general scheme showing one approach to the solid phasesynthesis of compounds of the invention.

FIG. 2 is a general scheme showing a second approach to the solid phasesynthesis of compounds of the invention.

FIG. 3 shows the structures of representative peptide bond surrogatesand replacements for peptide residues.

FIGS. 4-6 show synthetic schemes for representative compounds of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The unique compounds of the invention combine four key elements thathave never previously been simultaneously investigated:

-   -   (1) Recognition potential of amino acid and other functionalized        side chains    -   (2) Conformational rigidity and resistance to degradation of the        macrocyclic ring    -   (3) Modified hydrogen-bonding and polarity, improved stability,        and modulated physical properties and recognition potential of        peptide bond surrogates which have proven potential in bioactive        substances    -   (4) Spatial control through the non-peptidic tether component

The invention also has significant advantages over the existingcompounds and libraries for drug discovery:

-   -   dramatic improvement in information obtained from testing of the        compound libraries since control of conformation through the        tether component provides immediate knowledge of bioactive        forms;    -   control of conformation allows defined variation, with different        tethers, in the display of those recognition elements to attain        the three-dimensional orientation for which ligand-target        interactions could be maximized (the tether moiety can actually        constrain the cyclic structure into a single low energy        conformation in some cases);    -   high biological relevance through the use of amino acid side        chains in the recognition elements (A_(i)) to mimic existing        molecular recognition processes between proteins, nucleic acids        and proteins, or peptides and proteins;    -   applicability to broad range of targets and pharmaceutically        interesting biological systems, many of which have so far been        intractable for the development of small molecule        pharmaceuticals, such as those involving protein-protein or        protein-nucleic acid interactions;    -   utility of the compounds as probes for new and existing protein,        enzyme and receptor targets, with particular relevance given the        many new targets arising from the efforts in genomics and        proteomics;    -   increased speed in both hit follow-up and lead optimization of        bioactive molecules due to basic chemistry assembly method        remaining the same, with variations introduced primarily through        the individual building units, which can be readily used to        modulate and optimize any observed activity;    -   significant chemical diversity achieved through individual        building units that are designed to provide enhanced potential        for bioactivity    -   in the solid phase process for synthesis of these compounds, use        of a cleavage-release strategy from resin directly provides        compounds biased towards high purity as non-cyclic compounds        remain bound to solid support, thereby circumventing the usually        lengthy purification process; and    -   synthetic methods lead to a high degree of synthesis success        (>95%) with the ability to be straightforwardly scaled up for        larger material quantities since the original methodology was        developed as a standard solution phase process.

As such, the compounds of the invention are novel structures with highpotential in the search for new bioactive substances with favorableproperties.

Accordingly, the invention provides macrocyclic compounds of formulas(I) and (II)

wherein A₁, A₂, A₃, A₄, X, T and T₂ are as defined previously.

In a preferred embodiment of the macrocyclic compounds of formula (I), Tis chosen from the chosen from the group consisting of:

wherein the wavy line indicates a E, Z or a mixture of E and Z doublebond configuration;M₁, M₂, M₃, M₄ and M₅ are independently selected from O, S or NR₁₈wherein R₁₈ is selected from the group consisting of hydrogen, alkyl,substituted alkyl, formyl, acyl and sulfonyl;f₁, f₂, f₄, f₇ and f₁₀ are independently selected from 1, 2, 3 or 4;f₃ and f₈ are independently selected from 2 or 3;f₅, f₁₁, f₁₃, f₁₄ and f₁₅ are independently selected from 1 or 2;f₆ is 0, 1, 2, 3 or 4; andf₉ is 0, 1 or 2;(X) indicates the point of attachment of T to X; and(W) indicates the point of attachment of T to A₂, A₃ or A₄.

In a specific embodiment of the compound of formula (I), T is chosenfrom the group consisting of:

In a preferred embodiment of the macrocyclic compounds of formula (III),T₂ is chosen from the chosen from the group consisting of:

wherein the wavy lines indicate either a (R) or (S) stereochemistry ormixture thereof;q₁, q₂, q₃, q₆, q₇, q₈, q₉, q₁₀, q₁₁, q₁₃, q₁₅ and q₁₆ are eachindependently 1, 2, 3, 4 or 5;q₄ and q₁₈ are independently 1 or 2;q₁₂ and q₁₄ are each independently 0, 1, 2, 3 or 4;q₁₇ is 0, 1, 2 or 3;P₁, P₃ P₄ and P₅ are each independently O, S or NH;

P₆ is N or CH; P₇ is O or CR₅₂R₅₃;

R₃₆ is hydrogen, C₁-C₆ alkyl, benzyl or acyl;R₅₀ and R₅₁ are independently selected from the group consisting ofhydrogen, alkyl, hydroxy, alkoxy, and amino with the proviso that if oneof R₅₀ or R₅₁ is hydroxy, alkoxy or amino, the other is hydrogen oralkyl;R₅₂ and R₅₃ are independently selected from the group consisting ofhydrogen, alkyl, hydroxy, alkoxy, and amino with the proviso that if oneof R₅₂ or R₅₃ is hydroxyl, alkoxy or amino, the other is hydrogen oralkyl;R₅₄, R₅₅, R₅₆, R₅₇ and R₅₈ are independently selected from the groupconsisting of hydrogen, alkyl, hydroxy, alkoxy, and amino;R_(AA) is the side chain of a natural amino acid;(X) indicates the point of attachment of T to X; and(W) indicates the point of attachment of T to A₂, A₃ or A₄.

In yet another specific embodiment of the compound of formula (III), T₂is chosen from the group consisting of:

wherein Y is selected from hydrogen, alkyl, benzyl or acyl

The invention also provides compounds of formula (I) and formula (III)which are agonists or antagonists of a mammalian motilin receptor and/ora mammalian ghrelin receptor.

Motilin, a linear 22-amino acid peptide, plays a critical regulatoryrole in the GI physiological system through governing of fastinggastrointestinal motor activity. As such, the peptide is periodicallyreleased from the duodenal mucosa during fasting in mammals, includinghumans. More precisely, motilin exerts a powerful effect on gastricmotility through the contraction of gastrointestinal smooth muscle tostimulate gastric emptying, decrease intestinal transit time andinitiate phase III of the migrating motor complex in the small bowel.Due to the critical and direct involvement of motilin in control ofgastric motility, agents that either diminish (hypomotility) or enhance(hypermotility) the activity at the motilin receptor, are a particularlyattractive area for further investigation in the search for neweffective pharmaceuticals towards these indications. Macrocyclicantagonists of the motilin receptor are disclosed in U.S. Prov. Pat.Appl. Ser. No. 60/479,223.

Likewise, ghrelin is a key peptide hormone involved in a number ofimportant physiological functions including growth hormone secretion,maintenance of energy balance, appetite and gut motility. As such,antagonists of this receptor have been investigated for treatment ofobesity, while ghrelin agonists have interest in treatment of a varietyof diseases, including conditions caused by growth hormone deficiency,wasting syndrome, and GI disorders involving dysmotility.

(human, porcine, SEQ ID NO: 1)F-V-P-I-F-T-Y-G-E-L-Q-R-M-Q-E-K-E-R-N-K-G-Q motilin (human, SEQ ID NO:2) G-S-S(Oct)-F-L-S-P-E-H-Q-R-V-Q-Q-R-K-E-S-K-K-P-P- A-K-L-Q-P-R ghrelin

EXAMPLES

Table 1A and 1 B give the structure of 69 compounds according to theinvention.

Table 2 lists the mass spectral analytical data for these 69 compounds.

TABLE 1A Representative Compounds of formula (I) (I)

Compound A₁ A₂ A₃ T 101

T9 102

T9 103

T9 104

T9 105

T9 106

T9 107

T9 108

T9 109

T9 110

T9 111

T9 112

T9 113

T61 114

T54 115

T9 116

T9 117

T9 118

T9 119

T9 120

T50 123

T53 124

T9 125

T9 126

T9 127

T9 128

T9 129

T9 130

T9 131

T9 132

T9 133

T9 134

T9 135

T9 136

T9 137

T9 138

T9 139

T9 140

T45 141

T45 145

T48 148

T50 149

T10 151

T18 153

T2 154

T17 155

T1 156

T3 157

T16 158

T4 159

T5 163

T8 164

T9 165

T8 166

T8 167

T9 168

T9 169

T9 170

T9 X is NH, except for compound 167 where X is NMe and compound 168where X is NAc

TABLE 1B Representative Compounds of formula (III) (III)

Compound A₁ A₂ A₃ T₂ 121

T51 122

T51 142

T46 143

T47 144

T47 146

T49 147

T49 150

T21 152

T24 160

T12 161

T27 162

T14 X is NH

TABLE 2 Mass Spectral Analysis of Representative Compounds of theInvention Molecular Molecular Monoisotopic MS Found Compound FormulaWeight Mass (M + H)⁺ 101 C30H43N6O3Cl 571.2 570 571 102 C31H45N4O3Cl557.2 556 557 103 C31H44N3O3Cl 542.2 541 542 104 C31H42N3O3Cl 540.1 539540 105 C32H45N3O4 535.7 535 536 106 C30H44N4O4 524.7 524 525 107C30H44N4O4 524.7 524 525 108 C32H45N3O4 535.7 535 536 109 C32H47N3O4537.7 537 538 110 C32H47N3O4 537.7 537 538 111 C34H43N3O5 573.7 573 574112 C31H43N3O6 553.7 553 554 113 C32H39N5O3 541.7 541 542 114 C32H39N5O3541.7 541 542 115 C30H41N3O6 539.7 539 540 116 C32H46N4O5 566.7 566 567117 C31H44N4O5 552.7 552 553 118 C30H41N3O6 539.7 539 540 119 C30H41N3O6539.7 539 540 120 C27H41N3O5 487.6 487 488 121 C27H43N3O5 489.6 489 490122 C26H39N3O6 489.6 489 490 123 C26H41N3O6 491.6 491 492 124 C26H34N4O4466.6 466 467 125 C28H36N4O6 524.6 524 525 126 C25H32N4O4 452.5 452 453127 C29H41N5O4 523.7 523 524 128 C32H38N4O4 542.7 542 543 129 C26H34N4O5482.6 482 483 130 C32H46N4O4 550.7 550 551 131 C32H46N4O4 550.7 550 551132 C32H46N4O4 550.7 550 551 133 C32H46N4O4 550.7 550 551 134 C33H48N4O4564.8 564 565 135 C33H48N4O4 564.8 564 565 136 C33H48N4O4 564.8 564 565137 C33H48N4O4 564.8 564 565 138 C33H48N4O4 564.8 564 565 139 C29H40N4O4508.7 508 509 140 C24H35N4O5F 478.6 478 479 141 C24H35N4O5F 478.6 478479 142 C23H35N4O3F 434.5 434 435 143 C25H39N4O3F 462.6 462 463 144C25H39N4O3F 462.6 462 463 145 C26H40N4O3 456.6 456 457 146 C26H42N4O3458.6 458 459 147 C26H42N4O3 458.6 458 459 148 C27H42N4O3 470.6 470 471149 C28H35N4O5F 526.6 526 527 150 C24H33N4O6F 492.5 492 493 151C23H34N5O3F 447.5 447 448 152 C26H41N4O3F 476.6 476 477 153 C24H35N4O3F446.6 446 447 154 C23H31N4O3F 430.5 430 431 155 C21H32N4O4 404.5 404 405156 C23H32N4O3 412.5 412 413 157 C23H34N4O3 414.5 414 415 158 C25H32N4O3436.5 436 437 159 C26H34N4O3 450.6 450 451 160 C31H36N4O3S 544.7 544 545161 C23H34N4O4 430.5 430 431 162 C22H29N5O3S 443.6 443 444 163C29H41FN4O3 512.7 512 513 164 C30H43N4O3F 526.7 526 527 165 C29H39N5O4521.7 521 522 166 C29H39N5O4 521.7 521 522 167 C30H42N4O4 522.7 522 523168 C31H42N4O5 550.7 550 551 169 C28H38N4O4 494.6 494 495 170 C28H38N4O4494.6 494 495 Notes 1. Molecular formulas and molecular weights (MW) arecalculated automatically from the structure via ActivityBase software(IDBS, Guildford, Surrey, UK) or, for MW only, from the freeware programMolecular Weight Calculator v. 6.32 2. M + H obtained from LC-MSanalysis 3. All analyses conducted on material after preparativepurification

Synthesis Method

Building blocks for the construction of the compounds of the presentinvention include amino acids, hydroxyl acids, structures forincorporation of the peptide bond surrogates, and the tethers. Amino andhydroxyl acids are available commercially or synthesized via knownprocedures. Methods for construction of appropriate building blocks forthe peptide surrogates also are established in the art. (Mini-Rev. Med.Chem. 2002, 2, 463-473; Mini-Rev. Med. Chem. 2002, 2, 447-462; Curr.Med. Chem. 2002, 9, 2209-2229; Curr. Med. Chem. 2002, 9, 2243-2270;Curr. Med. Chem. 2002, 9, 963-978; Curr. Opin. Chem. Biol. 2002, 6,872-877; Curr. Opin. Chem. Biol. 1998, 2, 441-452; Angew. Chem. Int. Ed.Engl. 1994, 33, 1699; J. Med. Chem. 1993, 36, 3039-3049; J. Org. Chem.2000, 65, 7667-7675). Synthesis of the specific tether components havebeen described in WO 01/25257 and U.S. Prov. Pat. Appl. Ser. No.60/491,248.

An assortment of synthetic strategies can be used to access themacrocyclic compounds of the invention, several of which have alreadybeen disclosed in WO 01/25257. or are known in the literature.

An outline of a first preferred approach to the solid phase synthesis ofthe compounds of the invention, using a thioester strategy is providedin FIG. 1. A second preferred approach, called ring-closing metathesis(RCM), is also generally outlined in FIG. 2. Yet another alternativeapproach, utilizing an activated resin for cyclization, is presented inExamples 3 and 4.

Example 1 Representative Synthesis of Macrocyclic Compound of Formula IContaining Peptide Surrogate S6

The synthetic scheme is presented as FIG. 4( a). Starting frompolystyrene trityl resin containing the linker shown (1-0), initialstandard Fmoc deprotection of the linker furnished 1-1. This was thencoupled with Fmoc Nva-OH under standard conditions. Loading of the resinbased upon quantitative analysis of a cleaved aliquot at this stage was62%. After Fmoc cleavage using standard conditions followed by couplingwith Fmoc-(D)Val-OH under standard condition, 1-2 was obtained. AfterFmoc group deprotection, reductive alkylation with Fmoc-(D)Tyr(OtBu)-CHO1-3 was carried out as described in Step 1-d.

Step 1-d: Reductive Amination for Introduction of First Building Block

The following stock solutions were prepared first.

Solution A: 100 mL of 1% AcOH in TMOF (trimethylorthoformate).

Solution B: 100 mL of 1% AcOH in DMF/TMOF/MeOH (1:1:1).

After deprotection of the Fmoc group of 1-2 with 20% piperidine in DMF,2.0 g (1.5 mmol) of resin were washed 6 times with solution A, dried andtransferred into a 100 mL round bottom flask under a N₂ atmosphere.Next, 4.6 g (10.5 mmol, 7.0 eq) of aldehyde 1-3 (prepared from theWeinreb amide by LAH reduction using standard methods) were dissolved in25 mL of solution B and added to the resin. The mixture was stirred at50° C. for 45 min. To the above mixture was added 1.0 g (15.8 mmol, 10.5eq) of NaBH₃CN dissolved in 10 mL of solution B. The contents werestirred for an additional 2.5 h at 50° C. The resin was then washed withDMF (5×), then with alternate cycles of DCM/MeOH (2×), DCM (5×) anddried in vacuo.

Step 1-e: Cbz protection

To the above resin (1.5 mmol) was added 50 mL of DMF (DriSolv® (EMDChemicals, Inc., part of Merck KGaA, Darmstadt, Germanv) grade),followed by 4.0 mL (23 mmol, 15 eq) of DIPEA and 2.1 mL (15 mmol, 10 eq)of CbzCl. The mixture was agitated on an orbital shaker O/N. The resinwas then washed with DMF (5×), alternate cycles of DCM/MeOH (4×), DCM(5×) and dried in vacuo.

Step 1-f: Introduction of Partial Tether Component Via ReductiveAmination

After cleavage of the Fmoc group using standard conditions, 1.73 g (1.2mmol) of resin 1-4 was washed with 1% AcOH in MeOH (5×). To the resinwas then added a solution of 300 mg (1.8 mmol, 1.5 eq) of 1-5 in 15 mLof MeOH (DriSolv®) and 5 mL of TMOF. This was followed by addition of0.24 mL (2.4 mmol, 2.0 eq) of NaBH₃CN (or BAP) and the reaction was kepton the orbital shaker for 40 h due to the low solubility of 1-5. Theresin was washed with MeOH (10×), DMF/MeOH alternate cycles (5×),THF/MeOH alternate cycles (3×), THF (2×), DCM/MeOH alternate cycles(3×), CHCl₃ (2×), DCM (4×), then dried in vacuo.

To 1.7 g (1.2 mmol) of the above resin was added 30 mL of DMF (DriSolv®)followed by 2.8 mL (16 mmol, 13 eq) DIPEA and 1.7 mL (12 mmol, 10 eq) ofCbzCl. The mixture was agitated O/N. The resin was washed with DMF (5×),DCM/MeOH alternate cycles (3×) and DCM (5×), then dried in vacuo (oilpump). HPLC/MS analysis showed the desired product 1-6 to be formed.

Step 1-g: Macrocyclization via RCM

RCM was carried out with 1.2 g (0.84 mmol) of 1-6 following the StandardProcedure. Yield was 102 mg (24%) of the desired macrocycle (1-7) asdetermined by HPLC/MS/CLND analysis.

Step 1-h: Cbz and Unsaturation Hydrogenation

94 mg (0.11 mmol) of 1-7 was dissolved in 15 mL of glacial AcOH in a 50mL beaker and 188 mg of 10% Pd/C was added. After degassing, thesolution was stirred under 1000 psi of H₂ for 7 h. The reaction mixturewas then filtered through Celite® (World Minerals Inc., Santa Barbara,Calif.) and the Celite washed with 10 mL glacial AcOH (2×). The filtratewas then evaporated and the compound dried in vacuo. HPLC/MS analysisverified the identity of the product.

Step 1-i: tBu Group Deprotection

This step was carried out using 50% TFA:50% DCM:3% TIS(triisopropylsilane) for 2 h following standard methods. The crudematerial was purified by reverse phase preparative HPLC using MSdetection to trigger collection of the product (1-8).

Example 2 Representative Synthesis of Macrocyclic Compound of Formula IContaining Peptide Surrogate S6

The synthetic scheme is presented as FIG. 4( b). Anchoring ofFmoc-Nva-OH as well as subsequent Fmoc deprotection were performedfollowing standard procedures. The aldehyde 2-2 from Fmoc-(D)Val-OH wasprepared in 62% yield using standard methods. Reductive amination wasperformed as in step 1-d. Cbz protection of the resulting product underthe conditions described in step 1-e (repeated 2×) furnished the desiredproduct 2-3.

Fmoc deprotection, coupling with Bts-(D)Tyr(OtBu)-OH, and Mitsunobureaction of the resin bound tripeptides surrogate with 2-4 were allcarried out under standard conditions to give 2-5. Macrocyclization viaRCM with Hoveyda-Grubbs catalyst following the standard procedurefurnished the desired product, 2-6. CLND yield: 16.1 mg. Standarddeprotection of the Bts group is preferentially performed prior todeprotection of the Cbz group, with simultaneous reduction of the doublebond. The final product 2-7 is obtained by deprotection of the tBugroup.

Peptide Surrogates S16 or S17

The route presented here offers an alternative route to compounds of theinvention as illustrated for the general macrocyclic structure below.FIG. 5 details the reaction sequence as applied to a representativemacrocycle containing peptide surrogate S16.

Step 3-a: Standard Procedure for Loading Amino Acids to 2-ChlorotritylChloride Resin

In a 50 mL solid phase reactor, 2-chlorotrityl chloride resin (2 g, 2mmol/g, 4 mmol) was suspended in DCM (30 mL) and agitated for 15 min.After filtration, a solution of Fmoc-amino acid (4 mmol, 1 eq) and DIPEA(1.75 mL, 10 mmol, 2.5 eq) in DCM (15 mL) was added to the reactor andshaken for 2 h. The resin was filtered and washed with DMF (2×25 mL).(Optionally, but preferably, any remaining active sites on2-chlorotrityl chloride resin are then capped as follows.) The resinthus obtained was treated with a mixture (25 mL) of DCM:MeOH:DIPEA(80:15:5) for a period of 15 min and then filtered. This treatment wasrepeated once and the resin finally washed with DMF (3×25 mL) and driedin the standard manner.

Step 3-b: Standard Procedure for Deprotection of Fmoc Protective Groups

The resin from step 3-a was treated with a solution of piperidine in DMF(20%, v/v, 25 mL), agitated for 5 min, then filtered. This process wasrepeated once, except for 20 min, and the resin finally washedsuccessively with DMF (2×25 mL), iPrOH (2×25 mL), DMF (2×25 mL) and DCM(2×25 mL).

Step 3-c1: Coupling the Fmoc-Protected p-Nitrophenylcarbonate orp-Nitrophenylcarbamate (BB2)

A solution containing the Fmoc-protected amino alcohol ormono-Fmoc-protected diamine derivative (BB₂, 0.84 mmol, 4 eq) and HOBt(141 mg, 0.92 mmol, 4.4 eq) in DMF (4 ml) was added to the resin (410mg, 0.21 mmol) from Step A-2. To this suspension, DIPEA (366 μL, 2.1mmol, 10 eq) was added and the resulting mixture agitated for 12 h. Theresin was filtered, washed sequentially with DMF (2×5 mL), iPrOH (2×5mL), DMF (2×5 mL), iPrOH (2×5 mL) and DMF (3×5 mL), then dried in thestandard manner.

Step 3-c2: Standard Procedure for Coupling Amino Acids using the FmocProtection Strategy

In other embodiments, an amino acid could be desired in this position.In those instances, this procedure would eb employed. To a solutioncontaining the Fmoc (or Ddz)-protected amino acid (BB₂, 0.53 mmol, 2.5eq), and HOBt (121 mg, 0.79 mmol, 3.75 eq) in DMF (2.3 ml) was added DIC(94 μL, 0.58 mmol, 2.50 eq). This solution containing the now activatedamino acid was then added to the resin suspension (0.21 mmol, 210 mg)from Step A-2 and agitated for 3 h. The resin was filtered and washedsequentially with DMF (2×5 mL), iPrOH (2×5 mL), DMF (2×5 mL), iPrOH (2×5mL), DMF (3×5 mL), then dried in the standard manner.

Step 3-d: Removal of Fmoc Protective Group on BB₂

The resin was treated as described in step 3-cl, but at ⅕ of the scaletherein.

Step 3-e: Coupling of Bts-Amino Acid (BB₁)

A solution containing the Bts-amino acid (BB₁, 0.42 mmol, 2 eq), TBTU(202 mg, 0.63 mmol, 3 eq) and DIPEA (220 μL, 1.26 mmol, 6 eq) in DMF(2.5 mL) was added to the resin obtained in step 3-d and agitated for 3h. The resin was filtered and washed sequentially with DMF (2×5 mL),iPrOH (2×5 mL), DMF (2×5 mL), iPrOH (2×5 mL), DMF (3×5 mL), then driedin the standard manner.

Step 3-f: Mitsunobu Reaction

A solution containing the N-protected tether alcohol (0.84 mmol, 4 eq)and triphenylphosphine (220 mg, 0.84 mmol, 4 eq) in a mixture of toluene(2 mL) and tetrahydrofuran (2 mL) was added to the resin obtained instep 3-e. Finally, DIAD (166 μL, 0.84 mmol, 4 eq) was added and theresulting mixture was agitated for 12 h. The resin was filtered, washedsequentially with DMF (2×5 mL), iPrOH (2×5 mL), DCM (4×5 mL) and driedin the standard manner.

Step 3-g: Standard Procedure for Cleavage of the Protected MacrocyclicPrecursor from 2-Chlorotrityl Chloride Resin

The resin obtained from step 3-f was treated with a mixture of aceticacid:trifluoroethanol:DCM (1:1:8, v/v/v, 5 mL) for 2 h. The resin wasfiltered and washed once with a fresh 2.5 mL of the 1:1:8 mixture.Toluene (15 mL) was added to the filtrate and the solvents wereevaporated under reduced pressure. The alkylated tripeptide to serve asthe macrocyclic precursor was thus obtained, usually as a white solid.To confirm the amount priorto cleavage, an accurate weightofthe resinwas obtained in order to compare the weight gain observed with thequantity yielded from the cleavage.

Step 3-h: Standard Procedure for Loading Macrocyclic Precursor toActivated Resin

A solution containing the alkylated tripeptide (0.05 mmol) in DCM (5 mL)was added to the activated resin, for example TFP resin (213),o-nitrophenol resin (214), or o-chlorophenol resin (215) (300 mg). Inthis case, the latter was employed. Finally, DMAP (1 mg, 0.01 mmol, 0.2eq) and DIC (23 μL, 0.15 mmol, 3 eq) were added and the suspension wasagitated for 12 h. The resin was filtered, washed sequentially with DCM(2×5 mL), THF (2×5 mL), DCM (3×5 mL), then dried in the standard manner.

Step 3-i: Macrocyclization on Activated Resin

The resin obtained from step 3-h was treated with a solution of 2% TFA,3% TES (or TIPS) in DCM (v/v/v, 5 mL) for 30 min to remove the N-Ddzprotecting group of the tether element. The resin was filtered andwashed with DCM (2×5 mL). After Ddz deprotection, the resin was treatedwith a solution 2.5% DIPEA in THF (5 mL) for 1 h. The basicity of thesolution was confirmed during the reaction (wet pH paper) and more DIPEAadded if necessary to maintain basicity. The resin was filtered andrinsed with a fresh 2.5 mL of the 2.5% DIPEA in THF solution. Thecombined filtrate was evaporated under reduced pressure. Precipitationof the macrocycle was induced by adding H₂O to the residue. Themacrocycle was recovered by filtration and washed with H₂O to remove anyresidual salts. Alternatively, the residue was triturated with H₂O.

Step 3-j: The macrocycle obtained in step 3-i was subjected sequentiallyto the standard deprotection conditions to provide the final macrocycle.

Example 4 Representative Synthesis of Macrocyclic Compound of Formula IContaining Peptide Surrogate S3

This synthesis is presented in FIG. 6. The protocol highlights analternative method to those previously reported for introduction of thepeptoid moiety.

Step 4-a: In a 500 mL solid phase synthesis reactor was suspended2-chlorotrityl chloride resin (16.5 g, loading 2.0 mmol/g) in DCM (350mL). The resulting slurry was agitated for 30 min, filtered and washedwith DCM (2×350 mL). Separately, a solution of Bts-Gly-OH (13.4 g, 1.5eq) and DIPEA (17.2 mL, 3.0 eq) in DCM (350 mL) was prepared to form theBts-Gly salt. This solution of the carboxylate salt was added to theresin mixture and agitated for an additional 2.5 h. The reaction mixturewas filtered and the collected resin washed successively with DMF (3×350mL), 2-propanol (3×350 mL) and DCM (3×350 mL). Finally, any remainingactive sites on the resin were neutralized by treatment with a solutionof 85/10/5 DCM/MeOH/DIPEA (350 mL) for 1 h with agitation. The resultingresin was collected by filtration, washed successively with DMF (3×350mL), 2-propanol (3×350 mL) and DCM (3×350 mL) and dried under vacuum togive 18.73 g of 4-1.

Step 4-b: To the resin 4-1 (1.3 g) was added a solution of benzylalcohol (538 □I, 4.0 eq) and triphenylphosphine (1.40 g, 4.0 eq) in 10mL THF and 10 mL of toluene. The resin mixture was agitated for 1 minand then diisopropylazodicarboxylate (DIAD, 1.02 mL, 4.0 eq) is addedand agitation continued for 12 h. The resin was collected by filtration,washed successively with DMF (4×25 mL), 2-propanol (3×25 mL) and DCM(4×25 mL) and dried under vacuum to give 4-2.

Step 4-c: To the resin A2 was added a solution of mercaptoethanol (410μL, 10 eq) and n-propylamine (500 μL, 10 eq) in DMF (9 mL) and theresulting slurry agitated for 3 h. The resin was collected byfiltration, washed successively with DMF (3×25 mL), 2-propanol (3×25 mL)and DCM (3×25 mL) and dried under vacuum to provide 4-3.Step 4-d: To the resin 4-3 was added a solution of Bts-Gly-OH (695 mg,1.5 eq) and DEBPT (763 mg) in 9.4 mL of DMF. The resin mixture wasagitated for 1 min, then DIPEA (666 μL, 2.5 eq) is added and agitationcontinued for 3 h. The resin was collected by filtration, washedsuccessively with DMF (3×25 mL), 2-propanol (3×25 mL) and DCM (3×25 mL)and dried under vacuum to give 4-4.Step 4-e: To the resin 4-4 was added a solution of n-butanol (366 μL,4.0 eq) and triphenylphosphine (1.05 mg, 4.0 eq) in 10 mL THF and 10 mLof toluene. The resin mixture was agitated for 1 min, then DIAD (788 μL,4.0 eq) is added and agitation continued for 12 h. The resin wascollected by filtration, washed successively with DMF (4×25 mL),2-propanol (3×25 mL) and DCM (4×25 mL) and dried under vacuum to give4-5.Step 4-f: To the resin A5 was added a solution of mercaptoethanol (600μL, 10 eq), n-propylamine (500 μL, 10 eq) in DMF (6 mL) and theresulting slurry agitated for 3 h. The resin was collected byfiltration, washed successively with DMF (3×25 mL), 2-propanol (3×25 mL)and DCM (3×25 mL) and dried under vacuum to provide 4-6.Step 4-g: To the resin 4-6 was added a solution of Bts-Gly-OH (695 mg,1.5 eq) and DEBPT (763 mg) in 9.4 mL of DMF. The resin mixture wasagitated for 1 min, then DIPEA (666 μL, 2.5 eq) is added and agitationcontinued for 3 h. The resin was collected by filtration, washedsuccessively with DMF (3×25 mL), 2-propanol (3×25 mL) and DCM (3×25 mL)and dried under vacuum to give 4-7.Step 4-h: To the resin 4-7 was added a solution of Ddz-T1 (1.3 g, 4.0eq) and triphenylphosphine (1.05 g, 4.0 eq) in 10 mL THF and 10 mL oftoluene. The resin mixture was agitated for 1 min, then DIAD (788 μL,4.0 eq) is added and agitation continued for 12 h. The resin wascollected by filtration, washed successively with DMF (4×25 mL),2-propanol (3×25 mL) and DCM (4×25 mL) and dried under vacuum to give4-8.Step 4-i: To the resin 4-8 was added 10 mL of a solution of AcOH/TFE/DCM(1/1/8) and agitated for 2 h. The resin is filtered and washed with DCM(3×10 mL). The filtrate is evaporated to dryness and the residue furtherdried under high vacuum. The residual cleaved product is dissolved in 4mL of DCM, added to the 2-chlorophenol resin (450 mg), DIC (150 μL) andDMAP (15 mg) and agitated overnight. The resin is washed with DCM (3×),then dried under vacuum to give 4-9.Step 4-j: To the resin 4-9 was added 5 mL of a solution of 3% TFA in DCMand the resulting slurry agitated for 15 min. The resin is filtered, thetreatment repeated once, then the resin washed with DCM (3×5 mL) anddried under vacuum. To the dried resin was added 5 mL of a solution of2.5% DIPEA in THF and agitated for 1 h. The resin is filtered and washedwith THF (3×5 mL). The filtrate is evaporated under reduced pressure andthe residue dried under vacuum to yield the macrocycle 4-10.Step 4-k: The Bts group of 4-10 was removed using standard conditions toprovide the final, fully deprotected macrocycle, 4-11. This compoundcould be further purified by the standard methods.

Biological Evaluation for Compounds of the Invention

The compounds of the present invention were evaluated for their abilityto interact at the human motilin receptor and the human ghrelin receptorutilizing competitive radioligand binding assays as described in MethodB1 and B2, respectively. Further characterization of the interaction canbe performed utilizing the functional assays described in Methods B3 andB4 for the motilin and ghrelin receptors, respectively. All of thesemethods can be conducted, if so desired, in a high throughput mannertopermit the simultaneous evaluation of many compounds.

Results for the examination of representative compounds of the presentinvention using Methods B1 and B2 are presented in Table 3.

Example Method B1 Competitive Radioligand Binding Assay (MotilinReceptor) Materials:

-   -   Membranes were prepared from CHO cells stably transfected with        the human motilin receptor and utilized at a quantity of 1.5        μg/assay point. [PerkinElmer® SignalScreen® Product #6110544,        PerkinElmer, Inc., Wellesley, MAI    -   [¹²⁵I]-Motilin (PerkinElmer®, #NEX-378); final concentration:        0.04-0.06 nM    -   Motilin (Bachem®, #H-4385, Bachem AG Corporation, Bubendorf,        Switzerland); final concentration: 1 μM    -   Multiscreen Harvest plates-GF/B (Millipore®, #MAHFB1H60,        Millipore Corporation, Billerica, Mass.)    -   Deep-well polypropylene titer plate (Beckman Coulter®, #267006,        Beckman Coulter, Inc., Fullerton, Calif.)    -   TopSeal-A (PerkinElmer®, #6005185)    -   Bottom seal (Millipore®, #MATAHOP00)    -   MicroScint-0 (PerkinElmer®, #6013611)    -   Binding Buffer: 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 1 mM EDTA,        0.1% BSA

Assay Volumes:

-   -   150 μL of membranes diluted in binding buffer    -   10 μL of compound diluted in binding buffer    -   10 μL of radioligand ([125]-Motilin) diluted in binding buffer

Final Test Concentrations (N=11) for Compounds:

-   -   10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005 μM.

Compound Handling:

Compounds were provided frozen on dry ice at a stock concentration of 10mM diluted in 100% DMSO and stored at −20° C. until the day of testing.On the test day, compounds were allowed to thaw at room temperature andthan diluted in assay buffer according to the desired testconcentrations. Under these conditions, the maximum final DMSOconcentration in the assay was 0.5%.

Assay Protocol:

In deep-well plates, diluted cell membranes (1.5 μg/mL) are combinedwith 10 μL of either binding buffer (total binding, N=5), 1 μM motilin(non-specific binding, N=3) or the appropriate concentration of testcompound. The reaction is initiated by addition of 10 μl of[¹²⁵I]-motilin (final conc. 0.04-0.06 nM) to each well. Plates aresealed with TopSeal-A, vortexed gently and incubated at room temperaturefor 2 hours. The reaction is arrested by filtering samples throughpre-soaked (0.3% polyethyleneimine, 2 h) Multiscreen® Harvest platesusing a Tomtece Harvester (TomTec, Inc., Hamden Conn.), washed 9 timeswith 500 μL of cold 50 mM Tris-HCl (pH 7.4), and than plates areair-dried in a fumehood for 30 minutes. A bottom seal is applied to theplates prior to the addition of 25 μL of MicroScint™-0 to each well.Plates are than sealed with TopSeal-A and counted for 30 sec per well ona TopCount®D Microplate Scintillation and Luminescence Counter(PerkinElmer®) where results are expressed as counts per minute (cpm).

Data are analyzed by GraphPad Prism® (GraphPad Software, Inc., SanDiego, Calif.) using a variable slope non-linear regression analysis.K_(i) values were calculated using a K_(d) value of 0.16 nM for[¹²⁵I]-motilin (previously determined during membrane characterization).

$D_{\max} = {1 - {\frac{\begin{matrix}{{{test}\mspace{14mu} {concentration}\mspace{14mu} {with}\mspace{14mu} {maximal}\mspace{14mu} {displacement}} -} \\{{non}\text{-}{specific}\mspace{14mu} {binding}}\end{matrix}}{{{total}\mspace{14mu} {binding}} - {{non}\text{-}{specific}\mspace{14mu} {binding}}} \times 100}}$

where total and non-specific binding represent the cpm obtained in theabsence or presence of 1 μM motilin, respectively.

Example Method B2 Competitive Radioligand Binding Assay (GhrelinReceptor)

The competitive binding assay at the human growth hormone secretagoguereceptor (hGHS—R1a) was carried out analogously to assays described inthe literature. (Bednarek M A et al. (2000), Structure-function studieson the new growth hormone-releasing peptide ghrelin: minimal sequence ofghrelin necessary for activation of growth hormone secretagogue receptor1a; J Med Chem 43:4370-4376. Palucki B L et al. (2001),Spiro(indoline-3,4′-piperidine) growth hormone secretagogues as ghrelinmimetics; Bioorg Med Chem Lett 11:1955-1957.)

Materials

-   -   Membranes (GHS—R/HEK 293) were prepared from HEK-293 cells        stably transfected with the human ghrelin receptor (hGHS—R1a).        These membranes were provided by PerkinElmer® BioSignal®        (#RBHGHSM, lot#1887) (PerkinElmer BioSignal, Inc. Quebec,        Canada) and utilized at a quantity of 0.71 μg/assay point.    -   [¹²⁵I]-Ghrelin (PerkinElmer®, #NEX-388); final concentration:        0.0070-0.0085 nM    -   Ghrelin (Bachem®, #H-4864); final concentration: 1 μM    -   Multiscreen® Harvest plates-GF/C (Millipore®, #MAHFC1H60)    -   Deep-well polypropylene titer plate (Beckman Coulter®, #267006)    -   TopSeal-A (PerkinElmer®, #6005185)    -   Bottom seal (Millipore®, #MATAHOP00)    -   MicroScint™-0 (PerkinElmer, #6013611)    -   Binding Buffer: 25 mM Hepes (pH 7.4), 1 mM CaCl₂ 5 mM MgCl₂, 2.5        mM EDTA, 0.4% BSA

Assay Volumes

Competition experiments were performed in a 300 μL filtration assayformat.

-   -   220 μL of membranes diluted in binding buffer    -   40 μL of compound diluted in binding buffer    -   40 μL of radioligand ([¹²⁵I]-Ghrelin) diluted in binding buffer

Final test concentrations (N=1) for compounds of the present invention:

10, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005, 0.002, 0.001 μM. CompoundHandling

Compounds were provided frozen on dry ice at a stock concentration of 10mM diluted in 100% DMSO and stored at −80° C. until the day of testing.On the test day, compounds were allowed to thaw at rt overnight and thendiluted in assay buffer according to the desired test concentrations.Under these conditions, the maximal final DMSO concentration in theassay was 0.1%.

Assay Protocol

In deep-well plates, 220 μL of diluted cell membranes (finalconcentration: 0.71 μg/well) were combined with 40 μL of either bindingbuffer (total binding, N=5), 1 μM ghrelin (non-specific binding, N=3) orthe appropriate concentration of test compound (N=2 for each testconcentration). The reaction was initiated by addition of 40 μl of[1,1]-ghrelin (final conc. 0.0070-0.0085 nM) to each well. Plates weresealed with TopSeal-A, vortexed gently and incubated at rt for 30 min.The reaction was arrested by filtering samples through MultiscreenHarvest plates (pre-soaked in 0.5% polyethyleneimine) using a Tomtec®Harvester, washed 9 times with 500 μl of cold 50 mM Tris-HCl (pH 7.4, 4°C.), and then plates were air-dried in a fumehood for 30 min. A bottomseal was applied to the plates prior to the addition of 25 μL ofMicroScint-0 to each well. Plates were than sealed with TopSeal-A andcounted for 30 sec per well on a TopCount® Microplate Scintillation andLuminescence Counter (PerkinElmer®) using a count delay of 60 sec.Results were expressed as counts per minute (cpm).

Data were analyzed by GraphPad Prism (GraphPad Software, Inc., SanDiego, Calif.) using a variable slope non-linear regression analysis.K_(i) values were calculated using a K_(d) value of 0.01 nM for[¹²⁵I]-ghrelin (previously determined during membrane characterization).

D_(max) values were calculated using the following formula:

$D_{\max} = {1 - {\frac{\begin{matrix}{{{test}\mspace{14mu} {concentration}\mspace{14mu} {with}\mspace{14mu} {maximal}\mspace{14mu} {displacement}} -} \\{{non}\text{-}{specific}\mspace{14mu} {binding}}\end{matrix}}{{{total}\mspace{14mu} {binding}} - {{non}\text{-}{specific}\mspace{14mu} {binding}}} \times 100}}$

where total and non-specific binding represent the cpm obtained in theabsence or presence of 1 μM ghrelin, respectively.

Example Method B3 Aequorin Functional Assay (Motilin Receptor)Materials:

-   -   Membranes were prepared using AequoScreen® (EUROSCREEN SA,        Belgium) cell lines expressing the human motilin receptor (cell        line ES-380-A; receptor accession #AF034632). This cell line is        constructed by transfection of the human motilin receptor into        CHO-K1 cells co-expressing G_(α16) and the mitochondrially        targeted Aequorin (Ref #ES_WT-A5).    -   Motilin (Bachem®, #H-4385)    -   Assay buffer: DMEM-F12 (Dulbecco's Modified Eagles Medium) with        15 mM HEPES and 0.1% BSA (pH 7.0)    -   Coelenterazine (Molecular Probes®, Leiden, The Netherlands)

Final Test Concentrations (N=5) for Compounds:

-   -   10, 3.16, 1, 0.316, 0.1 μM.

Compound Handling:

Compounds were provided as dry films at a quantity of approximately 1.2μmol in pre-formatted 96-well plates. Compounds were dissolved in 100%DMSO at a concentration of 10 mM and stored at −20° C. until furtheruse. Daughter plates were prepared at a concentration of 500 μM in 30%DMSO with 0.1% BSA and stored at −20° C. until testing. On the test day,compounds were allowed to thaw at room temperature and than diluted inassay buffer according to the desired test concentrations. Under theseconditions, the maximum final DMSO concentration in the assay was 0.6%.

Cell Preparation:

Cells are collected from culture plates with Ca²⁺ and Mg²⁺-freephosphate buffered saline (PBS) supplemented with 5 mM EDTA, pelletedfor 2 minutes at 1000×g, resuspended in assay buffer (see above) at adensity of 5×10⁶ cells/mL and incubated overnight in the presence of 5μM coelenterazine. After loading, cells were diluted with assay bufferto a concentration of 5×10⁵ cells/mL.

Assay Protocol:

For agonist testing, 50 μl of the cell suspension was mixed with 50 μlof the appropriate concentration of test compound or motilin (referenceagonist) in 96-well plates (duplicate samples). The emission of lightresulting from receptor activation was recorded using the FunctionalDrug Screening System 6000 ‘FDSS 6000’ (Hamamatsu® Photonics K.K.,Japan).

For antagonist testing, an approximate EC80 concentration of motilin(i.e. 0.5 nM; 100 μL) was injected onto the cell suspension containingthe test compounds (duplicate samples) 15-30 minutes after the end ofagonist testing and the consequent emission of light resulting fromreceptor activation was measured as described in the paragraph above.

Results are expressed as Relative Light Units (RLU). Concentrationresponse curves were analyzed using GraphPad Prism® (GraphPad Software,Inc., San Diego, Calif.) by non-linear regression analysis (sigmoidaldose-response) based on the equation E=E_(max)/(1+EC₅₀/C)_(n) where E isthe measured RLU value at a given agonist concentration (C), E_(max) isthe maximal response, EC₅₀ is the concentration producing 50%stimulation and n is the slope index. For agonist testing, results foreach concentration of test compound were expressed as percent activationrelative to the signal induced by motilin at a concentration equal tothe EC₈₀ (i.e. 0.5 nM). For antagonist testing, results for eachconcentration of test compound were expressed as percent inhibitionrelative to the signal induced by motilin at a concentration equal tothe EC₈₀ (i.e. 0.5 nM).

Example Method B4 Aequorin Functional Assay (Ghrelin Receptor) Materials

-   -   Membranes were prepared using AequoScreen® (EUROSCREEN SA,        Belgium) cell lines expressing the human ghrelin receptor (cell        line ES-4,0-A; receptor accession #60179). This cell line is        constructed by transfection of the human ghrelin receptor into        CHO-K1 cells co-expressing G_(α16) and the mitochondrially        targeted Aequorin (Ref #ES_WT-A5).    -   Ghrelin (reference agonist; Bachem, #H-4864)    -   Assay buffer: DMEM (Dulbecco's Modified Eagles Medium)        containing 0.1% BSA (bovine serum albumin; pH 7.0).    -   Coelenterazine (Molecular Probes®, Leiden, The Netherlands)

Final test concentrations (N=8) for compounds of the invention:

10, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001 μM. Compound Handling

Stock solutions of compounds (10 mM in 100% DMSO) were provided frozenon dry ice and stored at −20° C. prior to use. From the stock solution,mother solutions were made at a concentration of 500 μM by 20-folddilution in 26% DMSO. Assay plates were then prepared by appropriatedilution in DMEM medium containing 0.1% BSA. Under these conditions, themaximal final DMSO concentration in the assay was <0.6%.

Cell Preparation

AequoScreen™ cells were collected from culture plates with Ca²⁺ andMg²⁺-free phosphate buffered saline (PBS) supplemented with 5 mM EDTA,pelleted for 2 min at 1000×g, re-suspended in DMEM—Ham's F12 containing0.1% BSA at a density of 5×10⁶ cells/mL, and incubated overnight at rtin the presence of 5 □M coelenterazine. After loading, cells werediluted with assay buffer to a concentration of 5×10⁵ cells/mL.

Assay Protocol

For agonist testing, 50 μL of the cell suspension was mixed with 50 μLof the appropriate concentration of test compound or ghrelin (referenceagonist) in 96-well plates (duplicate samples). Ghrelin (referenceagonist) was tested at several concentrations concurrently with the testcompounds in order to validate the experiment. The emission of lightresulting from receptor activation in response to ghrelin or testcompounds was recorded using the Hamamatsu® FDSS 6000 reader (Hamamatsu®Photonics K.K., Japan).

Analysis and Expression of Results

Results were expressed as Relative Light Units (RLU). Concentrationresponse curves were analyzed using GraphPad Prism® (GraphPad Software,Inc., San Diego, Calif.) by non-linear regression analysis (sigmoidaldose-response) based on the equation E=E_(max)/(1+EC₅₀/C)_(n) where Ewas the measured RLU value at a given agonist concentration (C), E_(max)was the maximal response, EC₅₀ was the concentration producing 50%stimulation and n was the slope index. For agonist testing, results foreach concentration of test compound are expressed as percent activationrelative to the signal induced by ghrelin at a concentration equal tothe EC₈₀ (i.e. 3.7 nM). EC₅₀, Hill slope and % E_(max) values arereported.

TABLE 3 Biological Activity of Representative Compounds of the InventionCompound Binding Activity [K_(i) (nM)]¹ Receptor² 101 A motilin (human)102 B motilin (human) 103 C motilin (human) 104 C motilin (human) 105 Cmotilin (human) 106 C motilin (human) 107 C motilin (human) 108 Cmotilin (human) 109 B motilin (human) 110 C motilin (human) 111 Bmotilin (human) 112 B motilin (human) 113 B motilin (human) 114 Bmotilin (human) 115 C motilin (human) 116 B motilin (human) 118 Bmotilin (human) 119 B motilin (human) 124 C ghrelin (human) 127 Cghrelin (human) 128 B ghrelin (human) 131 B ghrelin (human) 136 Cghrelin (human) 138 B ghrelin (human) 139 B ghrelin (human) 140 Cghrelin (human) 141 C ghrelin (human) 143 C ghrelin (human) 149 Bghrelin (human) 158 C ghrelin (human) 160 B ghrelin (human) 163 Cghrelin (human) 165 A ghrelin (human) 166 C ghrelin (human) 167 Bghrelin (human) 169 C ghrelin (human) ¹Activity presented indicated inthe following ranges: A = 0.01-0.10 μM, B = 0.1-1.0 μM, C = 1.0-10.0 μM²Binding conducted using Standard Methods described in the Examples

Although preferred embodiments of the present invention have beendescribed in detail herein and illustrated in the accompanying drawings,it is to be understood that the invention is not limited to theseprecise embodiments and that various changes and modifications may beeffected therein without departing from the scope or spirit of thepresent invention.

1-12. (canceled)
 13. A compound of formula (I): (I)

wherein A₄ is not present, A₃ is covalently bonded to T, and X, A₁, A₂,A₃ and T are selected from the group consisting of: X A₁ A₂ A₃ T NH

NH

NH

NH

NH

NH

N-Me

N-Ac

wherein (X) indicates the point of attachment of T to X and (A₃)indicates the point of attachment of T to A₃.
 14. An antagonist of amammalian motilin receptor having the structure:


15. An agonist of a mammalian ghrelin receptor having the structure: